Genes of the RAS family were found to be mutated in human cancers around 30 years ago, and the long term goal of this project is to develop a deeper understanding of the mechanisms by which these genes cause malignant transformation. The KRAS gene is the most commonly mutated family member, particularly in carcinomas of the pancreas, colon and lung. Together, these tumors account for hundreds of thousands of deaths worldwide each year. Using genetically engineered mouse models, this laboratory showed that a minor isoform of Kras (Kras4A) is essential for development of Kras mutant tumors. Mice deficient in this isoform are highly resistant to development of lung or skin tumors with Kras mutations, suggesting that inhibition of the function of Kras4A could result in therapeutic benefit for patients. This project involves a comprehensive analysis of the signaling pathway that is activated by this specific form of the Kras protein. A number of novel approaches will be used to understand how Kras4A signals within cells and tissues. A recently developed new imaging approach (Photo-activated localization microscopy, or PALM) will image the location of single molecules of Kras4A in cells, and find out whether there is an interaction between mutant and wild type Kras4A proteins. Signaling through canonical RAS pathway components in cells lacking Kras4A or both Kras4A and 4B, will be investigated in order to identify any specific signaling pathways that are deficient in these cells. Finally, an unbiased view of Kras signaling in vivo in mouse lungs will be developed, using novel gene expression network analysis tools. Comparison of gene expression networks from mice that are susceptible to lung cancer with those that are resistant due to deletion of Kras4A may identify pathways not previously associated with Ras that may account for the critical role of Kras4A in transformation. Using mouse models in vivo, the possible function of Kras4A in regeneration of lung epithelium from stem cells after damage will be studied, and new models in which Kras4A can be inhibited using inducible shRNA will be developed to investigate the role of this protein in the propagation and maintenance of tumors with mutant Kras genes.